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Biomedical Optics & Medical Imaging
Photoacoustic detection of circulating melanoma cells in human blood
Laser-induced ultrasound can aid the early detection and monitoring of metastatic disease.
22 May 2009, SPIE Newsroom. DOI: 10.1117/2.1200904.1630
Circulating melanoma cells (CMCs) spread in the blood and lymph systems, seeking to create secondary tumors at distant sites from the primary tumor.1–3 A patient with CMCs has metastatic disease and is in the most dangerous phase of the cancer cycle. Detection of CMCs may serve as an early indicator of metastasis or relapse, and tracking their number may enable noninvasive disease monitoring. Likewise, their absence may provide important staging information for clinicians and patients. Current methods, such as computed tomography scans and positron-emission tomography imaging, can only detect metastatic disease when secondary tumors are at least a few millimeters in size, and thus composed of millions or perhaps billions of cancer cells.
Detection of circulating tumor cells (CTCs) is an active research area that has met with limited success. Most methods, such as the real-time polymerase chain reaction (RT-PCR) technique, immunomagnetic separation, and fluorescence cytometry, use some sort of enhancement process to detect these cells or their biochemical precursors.4–6 These approaches are often time consuming, complex, and of limited accuracy. We have designed a rapid, label-free photoacoustic flowmetry system that can detect and count single melanoma cells in human blood samples (see Figure 1).
Figure 1. (top) Schematic of the photoacoustic flowmeter for detection of melanoma cells in blood samples. PVDF: Polyvinylidene fluoride. (bottom) Laser irradiating the glass flow chamber.
We used nanosecond laser pulses to irradiate white blood cells (WBCs) derived from patient blood samples and separated by standard centrifugation.7,8 If CMCs are present, they would reside among the WBCs because of their similar densities. Laser irradiation of pigmented CMCs induces distinct high-frequency acoustic transients. WBCs, on the other hand, do not have significant chromophores. By targeting melanin, a broadband optical absorber found in approximately 95% of melanoma cells, we exploit the high optical contrast of these cells among WBCs for specific detection.9 The rapid laser pulses induce heating and a thermoelastic expansion that results in a pressure wave originating from the optical absorber, the melanosomes within the melanoma cells. Once the optical energy is transduced into an acoustic pulse, the signal indicating the presence of CMCs propagates to an acoustic sensor in the flowmeter.
We tested the system on a cultured melanoma cell line and determined that our detection threshold was a single melanoma cell suspended in saline. To simulate realistic conditions, we tested the system on single melanoma cells spiked in WBCs. While the latter provide optical turbidity, they are acoustically transparent and did not inhibit CMC detection. We also tested our method on stage IV melanoma patients and found individual transient events corresponding to optically absorbing particles passing through the laser beam pulsing at 10Hz in the flow chamber (see Figure 2). We are designing a pilot study of stage IV patients using RT-PCR on tyrosinase, a melanin precursor, to co-register the photoacoustic signals we expect to find.
Figure 2. (left) Photoacoustic response from irradiating white blood cells from a healthy human volunteer. The signal at 2.2μs was constant and did not represent a particle inflow. (right) Response from a stage IV melanoma patient, showing transient signals corresponding to particles passing through the laser beam.
Although we tested the WBCs of healthy volunteers and did not find any photoacoustic signals after laser irradiation in the flowmeter, we anticipate that the only significant chromophore in the WBC layer of centrifuged blood can be hemoglobin from stray red blood cells (RBCs) that have been poorly separated from whole blood. We developed a process for near-simultaneous irradiation of the flow chamber with two laser wavelengths to classify RBCs and melanoma cells. Because the high absorption of hemoglobin in the blue and green contrasts with its low absorption at red wavelengths, photoacoustic signals from RBCs arising from irradiation at 433 and 630nm exhibit distinct differences from those arising from irradiating the relatively featureless absorption spectrum of melanin. We adapted a statistical scheme that relies on the mean and standard deviation of the ratio of the photoacoustic signals at both wavelengths.10 In addition, we used a Bayesian probit regression model that classifies melanoma from RBCs and also provides a measure of the classification uncertainty.
Our photoacoustic method provides rapid, inexpensive, and sensitive detection of CMCs as a means for finding and monitoring metastatic disease. This previously unavailable information will provide clinicians with a new tool to improve therapy for stage III and IV melanoma patients. We are also using this system to study the metastatic process for a deeper understanding of cancer and how it spreads. In addition, using specific tagging techniques, we are extending this approach to nonmelanoma cancers. By targeting cancer-specific receptors, such as HER-2 (human epidermal growth-factor receptor 2, a gene that helps control how cells grow, divide, and repair themselves) expression in breast-cancer cells, we can enhance a photoacoustic effect in otherwise invisible cancer cells by attaching exogenous chromophores. We have attached chromophores, such as dyed microspheres or nanoparticles (see Figure 3). By extending the reach of our photoacoustic flowmeter to breast, prostate, and other cancers, we may duplicate our success in melanoma detection for millions of cancer patients worldwide.
Figure 3. Scanning-electron micrograph showing attachment of 1μm black microspheres attached to a cultured breast-cancer cell from an MDA (malondialdehyde) cell line. These microspheres provide optical absorption for photoacoustic generation in an otherwise invisible target.
We acknowledge the Christopher S. Bond Life Sciences Center for support. We are grateful for funding from the Departments of Biological Engineering and Surgery, the Wallace H. Coulter Foundation, and the Missouri Life Sciences Trust Fund.
Department of Biological Engineering
University of Missouri
John A. Viator is an assistant professor of biological engineering and dermatology. His research interests focus on biomedical optics, particularly photoacoustic methods with applications in dermatology, oncology, and surgery.
4. K. Koyanagi, S. O'Day, R. Gonzalez, K. Lewis, W. Robinson, T. Amatruda, H. Wang, R. Elashoff, H. Takeuchi, N. Umetani, D. Hoon, Serial monitoring of circulating melanoma cells during neoadjuvent biochemotherapy for stage III melanoma: outcome prediction in a multicenter trial, J. Clin. Oncol. 23, no. 31, pp. 8057-8064, 2005.
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